Microflow Vessel Improving Reproducibility and Sensitivity of Electrochemical Measurements

A new microflow system was designed and developed for electrochemical measurements. The electrochemical electrodes prepared using thick film technology were used in this arrangement. Results of various measurements such as simple amperometric measurement on the example of H₂O₂ detection, measurement with glucose oxidase (GOx) biosensor, soluble enzyme activity measurement etc. carried out using this system are reported. It was observed that the sensitivity and reproducibility of the electrochemical measurements is improved significantly. The new device performance was proved on H₂O₂ detection, activity of GOx measurement and heavy metals detection (measured concentration range: H₂O₂ 10^?9 to 10^?1 M, glucose 10^?6 to 10^?2 M, activity of GOx 10^?1 to 10² IU, heavy metals (Cu, Pb) 10^?4 to 10^?3 M). The microflow insert greatly reduces the overall size of the electrolyte vessel and measurements with sample quantity as low as 2 mL can be accomplished.     

Electrochemical enzyme immunoassay using model labels

Enzyme immunoassays (EIAs) based on electrochemical detection offer several potential advantages and have been applied in clinical, medical, biotechnological, food and environmental analysis. Among the enzyme labels employed, horseradish peroxidase (HRP), alkaline phosphatase (ALP) and glucose oxidase (GOx) are the most common. This brief review reflects recent advances, challenges, and trends of electrochemical EIAs focusing on HRP, ALP or GOx as labels over the past five years. We especially emphasize current development of EIAs combined with other developments, including nanotechnology and miniaturization.     

Electrochemical Activity Studies of Glucose Oxidase (GOx)‐Based and Pyranose Oxidase (POx)‐Based Electrodes in Mesoporous Carbon: Toward Biosensor and Biofuel Cell Applications

A simple study using a fixed amount of mesoporous carbon (MSU‐F‐C) was performed for the comparison of pyranose oxidase (POx) and glucose oxidase (GOx) in their electrochemical performance under biosensor and biofuel cell operating modes. Even though the ratio of POx to GOx in the glucose oxidation activity per unit weight of MSU‐F‐C was 0.35, the ratios of POx to GOx in sensitivity and power density were reversed to be 6.2 and 1.4, respectively. POx with broad substrate specificity and an option of large scale production using recombinant E. coli has a great potential for various electrochemical applications, including biofuel cells.     

基于层-层自反应的葡萄糖氧化酶有序多层膜电极

以胱胺修饰的金电极为基础电极, 利用席夫碱反应使经高碘酸根氧化的葡萄糖氧化酶在该电极表面进行自身的层-层有序组装. 用电化学交流阻抗法对多层酶膜形成过程的跟踪结果表明, 该多层酶膜的生长是一个逐步形成的均匀过程. 用循环伏安法和I-t曲线法研究了该酶电极对葡萄糖的电催化氧化. 实验结果表明, 当采用羟基二茂铁作为人工电子转移媒介体时, 该酶电极对葡萄糖具有很好的电催化氧化功能. 该传感器制作简便, 响应迅速, 性能稳定, 催化电流与葡萄糖浓度在一定范围内成正比, 并且可以通过控制葡萄糖氧化酶的组装层数来调节该生物传感器的灵敏度与检测限.     

Study of ITO Glass Electrode Modified with Iron Oxide Nanoparticles and Nafion for Glucose Biosensor Application

In this study, we report the fabrication of the indium tin oxide (ITO) glass electrode modified with iron oxide nanoparticles (IONPs) and nafion for glucose biosensor applications. The IONPs was synthesized using the precipitation method and functionalized with citric acid (CA) to provide hydrophilic surface and functional group for glucose oxidase (GOx) enzyme immobilization. The structural and morphological studies of CA-IONPs were characterized using X-ray diffractometer (XRD) and transmission electron microscope (TEM). The size of the IONPs measured from TEM image was ∼17 nm. The bioelectrode designated as Nafion/GOx/CA-IONPs/ITO was developed by drop casting of the CA-IONPs, GOx and nafion on the ITO glass. The Nafion/GOx/CA-IONPs/ITO bioelectrode showed good electrochemical performance for glucose detection. The functionalized CA-IONPs acted as the catalyst and help to improve the electron transfer rate between GOx and ITO electrode. In addition, thin nafion film was coated on the electrode to prevent interference and improve chemical stability. The Nafion/GOx/CA-IONPs/ITO bioelectrode showed high sensitivity of 70.1 μAmM^-1cm^-2 for the linear range of 1.0-8.0 mM glucose concentrations.     

Electrochemical Behaviors of Glucose Oxidase Based on Biocatalytic Deposition of Gold Nanoparticles

Coupling nanotechnology with biocatalysis, a highly sensitive glucose biosensor for the study of electrochemical behaviors of glucose oxidase (GOx) was proposed by using monkshoodvine root–bark like carbon (MLC) as the platform for the biocatalytic deposition of AuNPs. The biosensor showed a linear range from 0.25 to 130 μM with a detection limit of 0.1 μM (S/N = 3) towards glucose and sensitivity of 3010 μA/mM. K value was calculated to be 67.4 μM. Furthermore, the proposed AuNPs/GOx–MLC modified pyrolytic graphite electrode (AuNPs/GOx–MLC/PGE) achieved direct electron transfer of GOx. Γ* was calculated to be 2.79 × 10^?11 mol/cm² and k_s was 1.79 s^?1. It also showed a remarkable electrocatalysis towards glucose.     

Evaluation of Enzymatic Kinetics of GOx‐based Electrodes by Scanning Electrochemical Microscopy at Redox Competition Mode

Glucose oxidase (GOx) is an enzyme, which is used for the development of enzymatic biofuel cells. Therefore in this research redox competition mode of scanning electrochemical microscopy (RC‐SECM) was applied for the investigation of glucose oxidase (GOx) catalyzed reaction kinetics. The GOx was immobilized by glutaraldehyde on substrates of different electrical conductivity: (i) gold covered glass was used as conducting substrate and (ii) plastic poly(methyl methacrylate) was used as non‐conducting substrate. Current vs distance dependencies were registered by SECM at different concentrations of glucose in the absence of redox mediator. The potential of −750 mV vs Ag/AgCl(3 M KCl) was applied to the microelectrode (ME), which was used as a probe in SECM, in order to register oxygen reduction current. Consumption of oxygen by the GOx based layer was evaluated according to principles determined by Michaelis‐Menten kinetics. Apparent Michaelis constants K _{M (app.)} were calculated from the dependencies of current vs glucose concentration. In both these cases the K _{M (app.)} value increased when the distance between ME and enzyme modified surface was increasing from 10 to 30 μm, while the K _{M (app.)} value decreased by increasing the distance from 30 to 60 μm.     

Electrochemical study of single wall carbon nanotubes/graphene/ferritin composite for biofuel cell applications

A single wall carbon nanotubes (SWNTs)/graphene/ferritin/GOx layer on a glassy carbon electrode (GCE) acting as a biofuel cell anode was fabricated using a SWNTs/graphene/ferritin composite as an electron transfer mediator from the enzyme to the electrode. In the presence of glucose, the SWNTs/graphene/ferritin/GOx composite showed a higher current response than SWNTs/graphene/GOx composite and the electrocatalytic oxidation of glucose on the anode increased linearly with increasing concentration of glucose. The highly distributed SWNTs/graphene/ferritin composite acts as a platform for enzyme immobilization resulted in an enhanced electrocatalytic activity towards glucose. The SWNTs/graphene/ferritin composite showed an enhanced electron transfer from enzyme to the electrode; therefore, SWNTs/graphene/ferritin/GOx composite can be used as an anode in biofuel cells.     

Multilayered construction of glucose oxidase on gold electrodes based on layer-by-layer covalent attachment

A feasible approach to construct multilayered enzyme film on the gold electrode surface for use as biosensing interface is described. The film was fabricated by alternate layer-by-layer deposition of periodate-oxidized glucose oxidase (GOx) and poly(allylamine) (PAA). The covalent attachment process was followed and confirmed by electrochemical impedance spectroscopy (EIS). X-ray diffraction (XRD) experiments revealed that the film was homogeneous and formed in an ordered manner with a thickness of 2.6 ± 0.1 nm per bilayer. The gold electrodes modified with the GOx/PAA multilayers showed excellent electrocatalytical response to the oxidation of glucose when ferrocenemethanol was used as an artificial redox mediator, which was studied by cyclic voltammetry (CV). From the analysis of voltammetric signals, the coverage of active enzyme on the electrode surface was estimated, which had a linear relationship with the number of GOx/PAA bilayers. This suggests that the analytical performance such as sensitivity, detection limit, and so on, is tunable by controlling the number of attached bilayers. The six GOx/PAA bilayer electrode exhibited a sensitivity of 15.1 μA mM⁻¹ cm⁻² with a detection limit of 3.8 × 10⁻⁶ M. In addition, the sensor exhibited good reproducibility and stability.     

扫描电化学显微镜直接电沉积法构建葡萄糖氧化酶微米点

利用自制的凹形电极在铂基底电极上直接构建了葡萄糖氧化酶微米点. 首先, 将电聚合和电化学刻蚀法相结合制备了凹形铂微米电极. 然后将此种电极作为参比及辅助电极, 基底铂电极作为工作电极, 利用葡萄糖氧化酶在合适的条件下(浓度、一定量Triton X-100存在、电极电位等)由于电极表面pH的降低可以在铂电极上电沉积这一特性, 将酶固定在铂基底电极上, 微修饰得到了具有活性的葡萄糖氧化酶微米点. 最终用扫描电子显微镜和扫描电化学显微镜对所得微米点进行了表征. 所得微米点直径约20 μm, 且具有催化活性. 该方法简便, 干扰因素较少.     

Nanoconfinement Effects: Glucose Oxidase Reaction Kinetics in Nanofluidics

Size‐tunable nanofluidic devices coupled to an electrochemical detector have been designed and then used to study glucose oxidase (GOx) reaction kinetics confined in nanospaces. The devices are fabricated via a photochemical decomposition reaction, which forms nanochannels covered with carboxyl groups. The generated carboxyl groups enable us to chemically pattern biological molecules on the polymer surfaces via covalent bonding. With this approach, the activity of the immobilized biological molecules confined in nanospaces with different sizes has been investigated. GOx species are chemically immobilized on the surface of the nanochannels, catalyzing the oxidation of substrate glucose as it flows through the channels. The enzyme reaction product, hydrogen peroxide, passing through the nanochannels, reaches an electrochemical detector, giving rise to an increase in anodic current. This steady‐state electrochemical current, which responds to various glucose concentrations, can be used to evaluate the GOx activity under confinement conditions. The results show significant nanoconfinement effects that are dependent on the channel size where the reaction occurs, demonstrating the importance of spatial confinement on the GOx reaction kinetics. The present approach provides an effective method for the study of enzyme activity and other bioassay systems, such as cell assays, drug discovery, and clinical diagnosis.     

Physicochemical Characteristics of Polypyrrole/(Glucose oxidase)/(Prussian Blue)‐based Biosensor Modified with Ni‐ and Co‐Hexacyanoferrates

In this work, three types of electrodes suitable for amperometric glucose biosensors were designed. One type of electrode was based on bio‐selective layer of polypyrrole/(glucose oxidase)/(Prussian Blue) (Ppy/GOx/PB) and it was used as a control electrode regarding to which electrochemical properties of two other types of electrodes were compared. During the formation of Prussian blue layers graphite electrodes were additionally modified by Ni‐hexacyanoferrate (NiHCF) and by Co‐hexacyanoferrate (CoHCF) in order to design Ppy/GOx/PB‐NiHCF and Ppy/GOx/PB‐CoHCF electrodes, respectively. Some physicochemical characteristics of all three types of electrodes were evaluated and compared. The Ppy/GOx/PB‐NiHCF electrode showed wider linear range of the calibration curve than Ppy/GOx/PB and Ppy/GOx/PB‐CoHCF electrodes. The effect of temperature on analytical performance of the Ppy/GOx/PB‐NiHCF based biosensor has been evaluated and activation energy of enzyme catalysed reaction has been calculated within the temperature range of 15 °C to 30 °C.     

Study of Factors Affecting Molecular Behaviors in Phenothiazine‐Mediated Biosensing by Electrochemical and Spectroscopic Methods

Reagentless glucose‐detecting biosensors were constructed by incorporating a series of phenothiazine derivatives as mediators onto chitosan (CHIT) matrix via different covalent bonds, wherein glucose oxidase (GOx) was employed as the enzyme. Electrochemical studies show significant decrease in the electrocatalytic current during cyclic voltammetric and amperometric measurements, resulting from complexes formation between GOx and phenothiazine molecules. This behavior was further verified by spectroscopic studies. The decrease in the peak intensity at 258 nm is due to the gradual complexes formation over time, consistent to the decrease in the current signal in electrochemical investigations. Correlation with the molecular structures of phenothiazine derivatives reveals a strong relationship between the hydrophobicity of the mediators and the stability of the biosensor electrodes.     

3,4,9,10‐Perylenetetracarboxylic Acid/Hemin Nanocomposites Act as Redox Probes and Electrocatalysts for Constructing a Pseudobienzyme‐Channeling Amplified Electrochemical Aptasensor

A simple wet‐chemical strategy for the synthesis of 3,4,9,10‐perylenetetracarboxylic acid (PTCA)/hemin nanocomposites through π–π interactions is demonstrated. Significantly, the hemin successfully conciliates PTCA redox activity with a pair of well‐defined redox peaks and intrinsic peroxidase‐like activity, which provides potential application of the PTCA self‐derived redox activity as redox probes. Additionally, PTCA/hemin nanocomposites exhibit a good membrane‐forming property, which not only avoids the conventional fussy process for redox probe immobilization, but also reduces the participation of the membrane materials that act as a barrier of electron transfer. On the basis of these unique properties, a pseudobienzyme‐channeling amplified electrochemical aptasensor is developed that is coupled with glucose oxidase (GOx) for thrombin detection by using PTCA/hemin nanocomposites as redox probes and electrocatalysts. With the addition of glucose to the electrolytic cell, the GOx on the aptasensor surface bioelectrocatalyzed the reduction of glucose to produce H₂O₂, which in turn was electrocatalyzed by the PTCA/hemin nanocomposites. Cascade schemes, in which an enzyme is catalytically linked to another enzyme, can produce signal amplification and therefore increase the biosensor sensitivity. As a result, a linear relationship for thrombin from 0.005 to 20 nM and a detection limit of 0.001 nM were obtained.     

Reduced Graphene Oxide/Pt Nanoparticles/Zn‐MOF‐74 Nanomaterial for a Glucose Biosensor Construction

In this study, a new glucose biosensor was fabricated by immobilizing glucose oxidase (GOx) on platinum nanoparticles (Pt NPs) decorated reduced graphene oxide (rGO)/Zn‐MOF‐74 hybrid nanomaterial. Herein, the biosensor fused the advantages of rGO with those of porous Zn‐MOF and conductive Pt NPs. This has not only enlarged the surface area and porosity for the efficient GOx immobilization and faster mass transport, but also provided favorable electrochemical features such as high current density, remarkable electron mobility through metal nanoparticles, and improved electron transfer between the components. The GOx‐rGO/Pt NPs@Zn‐MOF‐74 coated electrode displayed a linear measurement range for glucose from 0.006 to 6 mM, with a detection limit of 1.8 μM (S/N: 3) and sensitivity of 64.51 μA mM^?1 cm^?2. The amperometric response of the enzyme biosensor demonstrated the typical behavior of Michaelis‐Menten kinetics. The obtained satisfying sensitivity and measurement range enabled fast and accurate glucose measurement in cherry juice using the fabricated biosensor. The water‐stable Zn‐MOF‐74 demonstrated higher enzyme loading capacity and can be potent supporting material for biosensor construction.     

Immobilized Fullerene C60‐Enzyme‐Based Electrochemical Glucose Sensor

A mixed‐valence cluster of cobalt(II) hexacyanoferrate and fullerene C60‐enzyme‐based electrochemical glucose sensor was developed. A water insoluble fullerene C60‐glucose oxidase (C60‐GOD) was prepared and applied as an immobilized enzyme on a glassy carbon electrode with cobalt(II) hexacyanoferrate for analysis of glucose. The glucose in 0.1 M KCl/phosphate buffer solution at pH = 6 was measured with an applied electrode potential at 0.0 mV (vs Ag/AgCl reference electrode). The C60‐GOD‐based electrochemical glucose sensor exhibited efficient electro‐catalytic activity toward the liberated hydrogen peroxide and allowed cathodic detection of glucose. The C60‐GOD electrochemical glucose sensor also showed quite good selectivity to glucose with no interference from easily oxidizable biospecies, e.g. uric acid, ascorbic acid, cysteine, tyrosine, acetaminophen and galactose. The current of H₂O₂ reduced by cobalt(II) hexacyanoferrate was found to be proportional to the concentration of glucose in aqueous solutions. The immobilized C60‐GOD enzyme‐based glucose sensor exhibited a good linear response up to 8 mM glucose with a sensitivity of 5.60 × 10² nA/mM and a quite short response time of 5 sec. The C60‐GOD‐based glucose sensor also showed a good sensitivity with a detection limit of 1.6 × 10^‐6 M and a high reproducibility with a relative standard deviation (RSD) of 4.26%. Effects of pH and temperature on the responses of the immobilized C60‐GOD/cobalt(II) hexacyanoferrate‐based electrochemical glucose sensor were also studied and discussed.     

Optimization of Nanohybrid Biosensors Based on Electro-Crosslinked Tannic Acid Capped Nanoparticles/Enzyme

Enzymes/Nanoparticles (NPs) bioconjugates are massively used nowadays to develop thin films for optical and electrochemical biosensors. Nevertheless, their full characterization as a thin coating onto electrodes remains little discussed, in particular the influence of NPs size and enzyme/NPs ratio used in the electrodeposition solution. In this study, GOx (160 kDa) and HRP (44 kDa) were used in association with tannic acid capped gold NPs (a series with sizes from 7 to 40 nm) to electrodeposit biosensor coatings, sensitive towards glucose and H₂O₂, respectively. The electrodeposition process was based on a mussel-inspired electro-crosslinking between gallol moieties of tannic acid (at the surface of NPs) and amine moieties of the enzymes. On one hand, the sensitivity of the GOx/NPs coatings depends strongly on the NP size and the enzyme/NPs molar ratio of the electrodeposition solution. An optimal sensitivity was obtained by electrodeposition of 11 nm NPs at a GOx/NPs molar ratio close to the theoretical value of the enzyme monolayer. On the other hand, a modest influence of the NPs size was found on the sensitivity in the case of the electrodeposited HRP/NPs coatings, reaching a plateau at the HRP/NPs molar ratio close to the value of the theoretical enzyme monolayer. In both cases, the enzyme/NPs molar ratio played a role in the sensitivity. To fully understand the parameters driving the biosensor sensitivity, a comprehensive evaluation of the colloidal state of the bioconjugates is proposed here.     

Construction of glucose biosensor based on sorption of glucose oxidase onto multilayers of polyelectrolyte/nanoparticles

A new approach to constructing an enzyme-containing film on the surface of a gold electrode for use as a biosensor is described. A basic multilayer film (BMF) of (PDDA/GNPs) _n /PDDA was first constructed on the gold electrode by electrostatic layer-by-layer self-assembly of poly(diallyldimethylammonium chloride) (PDDA) and gold nanoparticles (GNPs). Glucose oxidase (GOx) was then sorbed into this BMF by dipping the BMF-modified electrode into a GOx solution. The assembly of the BMF was monitored and tested via UV-vis spectroscopy and cyclic voltammetry (CV). The ferrocenemethanol-mediated cyclic voltammograms obtained from the gold electrode modified with the (PDDA/GNPs) _n /PDDA/GOx indicated that the assembled GOx remained electrocatalytically active for the oxidation of glucose. Analysis of the voltammetric signals showed that the surface coverage of active enzyme was a linear function of the number of PDDA/GNPs bilayers. This result confirmed the penetration of GOx into the BMF and suggests that the BMF-based enzyme film forms in a uniform manner. Electrochemical impedance measurements revealed that the biosensor had a lower electron transfer resistance (R _et) than that of a sensor prepared by layer-by-layer assembly of PDDA and GOx, due to the presence of gold nanoparticles. The sensitivity of the biosensor for the determination of glucose, which could be controlled by adjusting the number of PDDA/GNPs bilayers, was investigated.     

Carbon Cavity Microelectrode for Electrical Wiring of Enzyme by Insoluble Electroactive Species in Aqueous Media

The carbon cavity microelectrode (CME), exhibiting a volume of 4×10^?6 cm³, offers a genuine alternative for immobilizing and connecting enzymes in aqueous electrolytes by powder of insoluble redox materials. In the present work, the electrochemical behavior of two redox species such as ferrocene (Fc) and tetrathiafulvalene (TTF) was investigated with CME to evaluate their potentialities in the electrical wiring of enzymes. For this purpose, powder of two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), was independently mixed with an insoluble redox material and forced to fill the single micro cavity of a carbon electrode covered by an inert insulator. The presence of the electroactive species, as well as the enzyme wiring was investigated by cyclic voltammetry. The amperometric detection of glucose was carried out by potentiostating the TTF/GOx and the Fc/GOx microelectrodes at 0.25 and 0.35 V respectively. The amperometric detection of H₂O₂ by the TTF/HRP microelectrode was performed at ?0.1 V vs. SCE.     

Glucose sensor based on redox-cycling between selectively modified and unmodified combs of carbon interdigitated array nanoelectrodes

We present a novel electrochemical glucose sensor employing an interdigitated array (IDA) of 1:1 aspect ratio carbon nanoelectrodes for the electrochemical-enzymatic redox cycling of redox species (ferricyanide/ferrocyanide) between glucose oxidase (GOx) and the two comb-shaped nanoelectrodes of the IDA. The carbon nanoelectrodes were fabricated using a simple, cost-effective, reproducible microfabrication technology known as the carbon-microelectromechanical-systems (C-MEMS) process. One comb (comb 1) of the IDA was selectively modified with GOx via the electrochemical reduction of an aryl diazonium salt, while the other comb (comb 2) remained unmodified; this facilitates electrochemically more active surface of comb 2, resulting in sensitive glucose detection. Ferricyanide is reduced to ferrocyanide by the GOx in the presence of glucose, and ferrocyanide diffuses to both combs of the IDA where it is oxidized. The limited electrochemical current collection at the surface-modified comb 1 is counterbalanced by the efficient redox cycling between the enzyme sites at comb 1 and the bare carbon surface of comb 2. Reducing the electrode-to-electrode gap between the two combs (gap = 1.9 μm) increases the diffusion flux of redox species at comb 2 hence, enhanced the sensitivity and limit of detection of the glucose sensor by ∼2.3 and ∼295 times, respectively at comb 2 compared to comb 1. The developed IDA-based glucose sensor demonstrated good amperometric response to glucose, affording two linear ranges from 0.001 to 1 mM and from 1 to 10 mM, with limits of detection of 0.4 and 61 μM and sensitivities of 823.2 and 70.0 μA mM⁻¹ cm⁻², respectively.